Methods for identifying RNA segments bound by RNA-binding proteins or ribonucleoprotein complexes

Abstract

The present invention relates to a method for identifying a binding site on an RNA transcript, wherein the binding site binds to one or more binding moieties. The method includes, among other things, introducing a photoreactive nucleoside into living cells wherein the living cells incorporate the photoreactive nucleoside into RNA transcripts during transcription thereby producing modified RNA transcripts; reverse transcribing the RNA of isolated cross-linked segments thereby generating cDNA transcripts with one mutation wherein the photoreactive nucleoside is transcribed to a mismatched deoxynucleoside; amplifying the cDNA transcripts thereby generating amplicons; and analyzing the sequences of the amplicons aligned against the reference sequence so as to identify the binding site, wherein the sequences of each amplicon having a mutation resulting from the introduction of the photoreactive nucleoside is considered to be a valid amplicon comprising at least a portion of a binding site on the RNA transcript.

What is claimed: 
     
       1. A method for identifying a binding site on an RNA transcript, wherein the binding site binds to one or more binding moieties, the method comprising
 a) introducing a 4-thiouridine photoreactive nucleoside into living cells wherein the living cells incorporate the photoreactive nucleoside into RNA transcripts during transcription thereby producing modified RNA transcripts; 
 b) irradiating said cells at a wavelength significantly absorbed by the photoreactive nucleoside to covalently cross-link a binding site on the modified RNA transcripts to one or more binding moieties; 
 c) removing all or part of the modified RNA transcripts which is not covalently cross-linked to the one or more binding moieties to form cross-linked segments having the photoreactive nucleoside, wherein the cross-linked segments comprise at least a portion of the binding site; 
 d) isolating the cross-linked segments thereby generating isolated cross-linked segments; 
 e) reverse transcribing the RNA of isolated cross-linked segments thereby generating cDNA transcripts with a deoxyguanosine (G) to deoxyadenosine (A) mutation wherein the photoreactive nucleoside is transcribed to a mismatched deoxynucleoside; 
 f) amplifying the cDNA transcripts thereby generating amplicons with a deoxythymidine (T) to deoxycytidine (C) mutation; 
 g) determining the nucleotide sequences of the amplicons having at least 15 nucleotides; 
 h) aligning the sequences of the amplicons against a reference sequence; and 
 i) analyzing the sequences of the amplicons aligned against the reference sequence so as to identify the binding site, wherein the sequences of each amplicon having the T to C mutation resulting from the introduction of the photoreactive nucleoside is considered to be a valid amplicon comprising at least a portion of a binding site on the RNA transcript. 
 
     
     
       2. The method according to  claim 1  further comprising removing the binding moiety from the isolated cross-linked segments thereby generating isolated segments prior to step (e). 
     
     
       3. The method of  claim 1  further comprising determining the sequence of a consensus motif, wherein the determination comprises using the mutation as an anchor and comparing the sequence surrounding the mutation to the reference sequence, wherein the mutation is within a sequence window that includes the mutation plus at least one nucleotide on either side of the mutation. 
     
     
       4. The method of  claim 3  wherein the sequence window includes five to twenty nucleotides on either side of the mutation. 
     
     
       5. The method of  claim 3  wherein the mutation is at the center of the sequence window. 
     
     
       6. The method according to  claim 1  wherein the reference sequence is a genomic sequence. 
     
     
       7. The method according to  claim 6  wherein the genomic sequence is a sequence that produced the RNA transcript. 
     
     
       8. The method according to  claim 1  wherein the reference sequence is a synthetic RNA sequence. 
     
     
       9. The method according to  claim 1  wherein the reference sequence is derived from an expressed sequence tag database. 
     
     
       10. The method according to  claim 1  further comprising identifying a feature required for interaction of the binding site and the binding moiety. 
     
     
       11. The method of  claim 1  wherein the binding moiety is a protein or a protein complex. 
     
     
       12. The method of  claim 11  wherein the protein complex comprises miRNA, piRNA, siRNA, endo-siRNA, snoRNA, snRNA, tRNA, rRNA or a combination thereof. 
     
     
       13. The method of  claim 11  wherein the protein is an RNA-binding protein, an RNA-associated protein or a combination thereof. 
     
     
       14. The method of  claim 1  wherein the binding site is a coding transcript. 
     
     
       15. The method of  claim 1  wherein the binding moiety is epitope-tagged. 
     
     
       16. The method of  claim 1  wherein step (c) comprises (i) forming a soluble extract of the cells; and (ii) treating the extract with a nuclease thereby forming the cross-linked segments. 
     
     
       17. The method of  claim 16  wherein the nuclease is ribonuclease-T1. 
     
     
       18. The method of  claim 17  wherein aligning the sequences of the amplicons comprises determining which amplicons have the characteristic that, when aligned with the reference sequence, the reference sequence has a guanosine one nucleotide upstream from the 5′ end of the amplicons. 
     
     
       19. The method of  claim 17  wherein analyzing the sequences of the amplicons comprises determining which amplicons have the characteristic that, when aligned with the genomic sequence, the genomic sequence has a guanosine one nucleotide upstream from the 5′ end of the amplicons, wherein such amplicons are valid amplicons. 
     
     
       20. The method of  claim 1  wherein aligning the sequences of the amplicons comprises determining which amplicons have a mutation wherein a deoxythymidine of the reference sequence is replaced by a deoxycytidine in the amplicons. 
     
     
       21. The method of  claim 1  wherein analyzing the sequences of the amplicons comprises determining which amplicons have only one mutation wherein a deoxythymidine of the reference sequence is replaced by a deoxycytidine in the amplicons, wherein such amplicons are valid amplicons. 
     
     
       22. The method of  claim 15  wherein isolating the cross-linked segments comprises immunoprecipitation. 
     
     
       23. The method of  claim 22  further comprising separating the cross-linked segments by length after immunoprecipitation. 
     
     
       24. The method of  claim 22  further comprising treating the cross-linked segments with nuclease after immunoprecipitation. 
     
     
       25. The method of  claim 1  wherein removing the binding moiety comprises digesting the binding moiety with a protease. 
     
     
       26. The method of  claim 1  wherein the wavelength is greater than 300 nm. 
     
     
       27. The method of  claim 1  wherein the living cell is part of a cell culture, a cell extract, whole tissue, or a whole organ.